Sheep Choroid Plexus Research Articles

Neutral amino acid uptake by the isolated perfused sheep choroid plexus.

1. The uptake of neutral amino acids from the blood into the cells of the choroid plexus was studied by means of the rapid (less than 40 s) single-circulation paired-tracer dilution technique in the isolated perfused choroid plexus of the sheep. 2. The study provides the first direct evidence for the carrier-mediated entry of neutral amino acids from blood into the cells of the choroid plexus. 3. In the terms of Christensen's classification the presence of L-amino acid carrier systems for large neutral amino acids with bulky side chains has been demonstrated. 4. No measurable uptake of [14C]methyl amino isobutyric acid ([14C]MeAIB) during a single passage through the choroid plexus circulation was demonstrated which indicates the probable absence of a significant 'A' transport system. 5. The uptake of small neutral amino acids such as glycine and L-alanine was shown to be carrier-mediated. Results suggest that these amino acids are mainly transported by the glycine and ASC carrier systems, respectively. 6. The results suggest that there is a similarity between the transport systems for neutral amino acids on the blood side of both the blood-brain barrier and blood-cerebrospinal fluid barrier, the exception being for the presence of a glycine carrier on the blood side of the choroid plexus.

Étude ultrastructurale du virus visna en culture de cellules de plexus choroide de mouton

Ultrastructural study on visna virus in sheep plexus choroid cells The Visna virus, a model for the study of Lentivirinae , was observed in cell culture from sheep choroid plexus. Ultrathin sections showed two steps of virus leaving the cells. The budding structures on the cell surface showed crescents of virus-like particles, enclosed by an envelope surrounded by spicules. Free virus particles in the extracellular medium presented different stages of virus maturation: the clear immature stage, the intermediate forms and the matured infectious virions. This progressive evolution explains the continuity in the different forms and stages of virus development, and the final formation of viral nucleotide enclosed by an internal membrane. Le virus Visna, modèle des Lentivirinae , a été observé en culture cellulaire de plexus choroïde de mouton. Les coupes ultrafines montrent les deux étapes de la sortie du virus. La constitution de bourgeons cellulaires apparaît comme une formation en croissant qui est entrouré d'une enveloppe, elle-même hérissée de spicules. Les virus libres dans le milieu extracellulaire subissent plusieurs stades de maturation: les particules claires, les formes intermédiaires et les virions mûrs infectieux. Cette évolution progressive met en évidence la filiation virale entre ces différentes formes dont l'aboutissement est l'identification du nucléoïdes entouré d'une membrane interne.

Transfer of magnesium across the perfused choroid plexus of sheep.

Isolated perfused choroid plexus preparations from sheep were used to study the effects of low concentrations of magnesium in the perfusion fluid on the transfer of magnesium into choroid plexus fluid (CPF). A perfusion fluid of similar electrolyte composition to sheep blood resulted in CPF similar to ventricular cerebrospinal fluid at a rate of 2.2 microliter min-1 mg-1 dry choroidal tissue. Decreasing the concentration of magnesium in the perfusion fluid caused a fall in the concentration of magnesium in the CPF, although it remained higher than in the perfusion fluid. The rate of transfer of magnesium from the perfusion fluid to the CPF decreased in the presence of high levels of potassium in the perfusion fluid. But decreasing the concentration of calcium in the perfusion fluid had no effect on magnesium transfer rates. These results suggest that the ability of the choroid plexus to transport magnesium against a concentration gradient is an important control of the concentration of the cerebrospinal fluid. However, this ability is insufficient to maintain cerebrospinal fluid concentrations of magnesium at normal levels when the blood magnesium concentration is below about 0.5 mmol l-1.

Reverse transcriptase activity in a case of ovine lymphoblastic leukaemia

Tissues taken post mortem from a sheep suffering from lymphoblastic leukaemia were examined for retroviral reverse transcriptase (RT) activity and for particles of retroviral density. Cultured bone marrow and lymph node cells were found to release into the medium particles of 1.16 and 1.21 g/ml density, and poly rC-oligo dG templated activity could be detected in cell-free supernatants. Tissues from two normal sheep were not found to exhibit these characteristics. Electron microscopic observation failed to reveal any retrovirus-like particles in the RT positive tissues. In vitro transmission studies indicated that the RT activity could not be transmitted to either sheep choroid plexus or lamb trachea cells, but was transmitted, at low efficiency, to both bovine kidney and mink lung cells.

Precursor polypeptides to structural proteins of visna virus.

Visna virus is a retrovirus which replicates in fibroblast-like cells of the sheep choroid plexus through a lytic cycle. Visna virions contain three major low-molecular-weight proteins (p30, p16, and p14) which, together with the genomic RNA and several molecules of reverse transcriptase, constitute the core structure of the virions. The core is surrounded by an envelope containing a major glycoprotein (gp135). By analogy with the oncoviruses, these three groups of structural proteins (i.e., the internal proteins, the envelope glycoprotein, and the reverse transcriptase) are probably encoded by the gag, env, and pol genes, respectively. To elucidate the genetic organization of the visna virus genome and its expression, we studied the synthesis of viral proteins in infected sheep choroid plexus cells. Intracellular viral proteins were detected by immunoprecipitation of pulse-labeled cell extracts with monospecific sera raised against p30, p16, and gp135 and resolution of the proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Immunoprecipitation with anti-p30 and anti-p16 sera allowed the characterization of the 55,000-dalton polypeptide precursor to internal virion proteins p30, p16, and p14 (Pr55(gag)). Tryptic peptide mapping confirmed the precursor-product relationship between Pr55(gag) and the three internal proteins. In addition, a gag-related polypeptide of 150,000 daltons was also detected. This polypeptide, which was less abundant than Pr55(gag), is a likely precursor to the viral reverse transcriptase (Pr150(gag-pol)). Pr55(gag) and Pr150(gag-pol) are not glycosylated. The precursor related to major envelope protein gp135 is a glycosylated polypeptide with an average molecular weight of 150,000 (gPr150(env)). Pulse-chase experiments indicated that gPr150(env) matures into glycoprotein gp135 intracellularly; however, gp135 was never preponderant in cell extracts. The non-glycosylated from of gPr150(env), which accumulated in the presence of 2-deoxy-d-glucose, appeared as a polypeptide of about 100,000 daltons. These results indicated that visna virus codes for the largest non-glycosylated env-related precursor among all of the retroviruses and therefore probably contains the largest env gene.

Intracellular ribonucleoprotein complexes of visna virus are infectious.

Sheep choroid plexus cells infected with visna virus produce intracytoplasmic viral ribonucleoprotein complexes with sedimentation values of 120S to 200S and buoyant densities of 1.29 to 1.32 g/cm3. These ribonucleoprotein complexes display an endogenous RNA-directed DNA polymerase activity and contain all of the species of RNA associated with polysomes. An analysis of the polypeptides present in the ribonucleoproteins allowed us to identify the mature internal virion core proteins and their precursor, Pr55gag, as well as the glycosylated envelope precursor gPr150env and small amounts of mature glycoprotein gp135. Ultracentrifugation-purified ribonucleoproteins could infect sheep choroid plexus cells and led to a normal lytic cycle with virus production. Our results suggest that visna virus can propagate by means of intracellular infectious particles.

Slow virus replication: the role of macrophages in the persistence and expression of visna viruses of sheep and goats.

Lentiviruses of sheep and goats cause slowly progressive diseases of the central nervous system (visna), lungs (maedi) and joints (arthritis) in their natural hosts. However, the virus target cell(s) in these diseases are still unknown. In this report, using laboratory-adapted Icelandic visna virus and several field strains recently obtained from sheep and goats with natural disease in the U.S.A., we show that macrophages became persistently infected when inoculated in culture. Furthermore, macrophages were an invariable source of virus from experimentally and naturally infected animals. Virus-producing macrophages developed minimal cytopathic changes and virus assembly occurred mainly intracellularly, accumulating in cytoplasmic vacuoles. In contrast to macrophages, sheep choroid plexus fibroblasts developed syncytial cytopathic changes after inoculation and virus maturation occurred at the cell surfaces. Replication of the Icelandic virus was highly productive in this system but that of the field viruses was very inefficient. In some cases these agents failed to replicate in the fibroblasts and no cytopathic effect occurred. This block in the field virus replication was, however, overcome when infected nonproducer fibroblasts were co-cultivated with macrophages. In these cases, virus production with attendant cytopathic effect in the fibroblasts required the continuous presence of macrophages because the cells reverted to a non-productive state when separated from macrophages and became productive again when subcultures were added to new macrophages. The roles of the macrophage as a virus target cell and virus inducer in the virus-macrophage-fibroblast interactions are discussed with inferences to the well-known phenomenon of restricted virus replication in infected animals and the immunopathological aspects of the diseases.

Wild-type temperature-sensitive and -resistant visna viruses: isolation and biological comparison.

The plaques formed by wild-type (WT) populations of strain K485 visna (V-K485) virus, strain K796 visna (V-K796) virus, and of progressive pneumonia virus in sheep choroid plexus (SCP) cell cultures are heterogeneous in size. Plaque purification procedures showed that this heterogeneity was due to the presence of two biologically different viruses, large plaque-forming (Lpf) virus and small plaque-forming (Spf) virus. The V-K796 Lpf virus, the V-K796 Spf virus, and the V-K796 WT virus are antigenically similar to each other. Although the V-K796 Spf virus is less cytopathic than the V-K796 Lpf virus, both viruses have a 14- to 16-hour latent period in SCP cells and have similar rates of synthesis for the initial 24 to 30 hours after infection. The V-K796 Spf virus is considered a temperature-sensitive visna virus since, unlike the V-K796 Lpf virus, it does not produce plaques at 41 degrees C and it is more thermosensitive than the V-K796 Lpf variant. Population analyses of two strains of wild-type visna virus and one strain of progressive pneumonia virus demonstrated that these populations contain variants with the phenotypes of V-K796 Spf virus and V-K796 Lpf virus, that the Spf virus was not selected against in sheep, and that cultivation of the wild type visna viruses at 37 degrees C selected against the temperature-sensitive variants. The existence of the temperature-sensitive Spf viruses in these wild-type virus populations suggests that the Spf variants maintain the persistent visna virus infection in sheep and delay the development of the host's cell-mediated immune response to the viral proteins.

Visna virus DNA: Discovery of a novel gapped structure

We have analyzed the structure of visna virus DNA synthesized during the life cycle of this lytic virus in confluent cultures of sheep choroid plexus cells. Viral DNA is of predominantly two forms: A linear duplex of about 6 million daltons corresponding to a transcript of viral RNA of subunit size; and, a structure with a gap in the center comprised of a full-length minus strand, and two long plus strands that together are about 300–500 bases shorter than the minus strand. A physical map of the linear DNA derived with restriction enzymes was ordered with respect to viral RNA, and the DNA was shown to bear terminal repeats of 350–450 nucleotides. Two circular forms of visna DNA differing only by the length of one terminal repeat also were detected, but constituted a very minor population. We discuss the relationship of these observations with this prototype of the lentiviruses to those recorded for transforming retroviruses.

Kinetics of maedi virus production in sheep choroid plexus cells

The growth characteristics of two lines of maedi virus were studied by measurements of released viral RNA, by reverse transcriptase assay and by infectivity assay. The measurements were made from sheep choroid plexus cell culture media collected at different time intervals during an incubation period of 2 weeks. The amount of virus was determined by measuring labelled RNA in culture fluids at 2-day intervals. The extracted virus-specific RNA formed uniform 60–70 S peaks in rate zonal sedimentation gradients. No definite signs of 30–35 S subunits were seen. A prominent increase in 60–70 S RNA production was seen between days 4 and 6 post-infection, after which the amount of viral RNA declined perceptibly. Some differences were observed between the growth curves of the maedi virus lines studied. These results were supported by reverse transcriptase assays. The highest reverse transcriptase activity was between days 4 and 6; for maedi 1320 it was a little earlier than for maedi M88. The activity was still quite high on the 11th day after infection. Virus release was also studied by serial titrations of infectivity. The titre increased conspicuously after 2 days of incubation and levelled off at about 4 days for maedi 1320 but reached a peak at about 6 days after infection for maedi M88.

Actin containing filaments in sheep choroid plexus cells infected in vitro by Visna virus: An immunofluorescent and ultrastructural study

The in vitro infection of sheep choroid plexus cells by Visna virus induces changes in the distribution and the orientation of actin containing filament bundles. During the cell fusion the stellate shape of cells with several interdigitations is associated with radial spanning of the contractile system. The intercellular contacts are characterized by the occurrence of a discontinuous line of rich actin containing dots which could be a junction of several short filament bundles.

Phosphonoformate inhibition of visna virus replication.

Phosphonoformate (PFA) inhibits multiplication of visna virus in sheep choroid plexus cells; a 50% reduction of virus yield was obtained by 20 to 80 microM PFA. Morphological changes, such as syncytial formation and cell degeneration, could be reversibly prevented by PFA. Cell growth was not significantly affected at 500 microM PFA, although prolonged treatment with 2 mM PFA did arrest cell growth. Cell-free reverse transcriptase activity primed with various synthetic template-primers was inhibited about 90% in the presence of 100 microM PFA. The results from kinetic experiments suggested that reverse transcriptase was utilized early but not late in the infection cycle. A structurally related substance, phosphonoacetate, did not inhibit visna virus multiplication and had no inhibitory effect on reverse transcriptase activity at a concentration of 500 microM.

One-cycle growth studies with Maedi—Progressive pneumonia—Visna viruses: Shorter latent periods revealed

The latent periods of Visna virus and of progressive pneumonia virus were demonstrated by one-cycle growth studies to end 14–16 hr after infection of sheep choroid plexus cell cultures. Maedi virus was demonstrated to have a 14–18-hr latent period.

Resistance of Visna Virus to Interferon

Visna is a slow infection of sheep caused by a retrovirus. The persistence of virus despite the immune response of the host is best explained by restricted genetic expression of the virus and consequently prolonged periods of residence inside cells. The purpose of this investigation was to determine whether the restriction in genetic expression of visna virus is mediated by interferon. Sheep interferon induced by polyriboinosinic-polyribocytidylic acid in fetal lambs inhibited the growth of herpes simplex virus, vesicular stomatitis virus, and vaccinia virus, but even highly concentrated interferon did not affect the replication of visna virus in sheep choroid plexus cells. The same results were obtained whether the effects of interferon were assessed in single of multiple cycles of growth and when interferon was added at later times in the growth cycle of the virus. This unusual resistance of visna virus to interferon suggests that restriction of viral expression by the host is probably not mediated in this way.

Morphological aspects of some sodium transporting epithelia suggesting a transcellular pathway via elements of endoplasmic reticulum

Electron microscopic studies of sodium transporting epithelia from frog skin, sheep choroid plexus, rabbit gallbladder and small intestine, and rat kidney revealed the presence of a complex intracellular system of tubulo-cisternal endoplasmic reticulum which appeared to connect apical (luminal) and baso-lateral cell surfaces. The system was present in the tight epithelium of frog skin but was most abundant in leaky epithelia with low transepithelial resistance and isotonic transport. The basic structural features of the system and its relationship with some associated components are described. Our result, coupled with preliminary physiological studies, indicate that developmental and seasonal (hormone-induced) changes in the configuration of the tubulo-cisternal endoplasmic reticulum may be closely correlated with specific changes in epithelial permeability. The findings are discussed in the light of the hypothesis that epithelia possess two sodium transporting systems: One based on pump sites in the plasma membrane producing a hypertonic transportate and another located in the membranes of the tubulo-cisternal endoplasmic reticulum which, due to its extensive surface, would be well suited for producing an isotonic transportate.

Characterization of visna virus envelope neuraminic acid

Visna virus particles inhibit influenza virus hemagglutination in an assay for neuraminic acid-containing viruses. Pretreatment of visna virus with neuraminidase abolished hemagglutination inhibition activity but did not significantly affect attachment, infectivity, or virus-induced cell fusion in sheep choroid plexus cell monolayers.

Ovine cells: Their long-term cultivation and susceptibility to visna virus

Sheep choroid plexus (SCP) cells have been subcultured more than 120 times and have undergone over 300 cell generations. These fibroblastic-appearing SCP II-B cells contain ovine-specific antigens, have an absolute plating efficiency of 23 to 28% and are as susceptible to visna virus infection and virus-induced cytopathology as their low passage level counterparts. Cultures of low, relatively high and high passage level SCP cells produced equivalent amounts of visna virus at similar rates when infected with equal amounts of visna virus. The passage level of the SCP II-B cells, their elapsed number of cell generations, their possession of ovine-specific antigens and their full susceptibility to visna virus allow these cells to be considered an established line of sheep cells.

Lack of correlation between tight junction morphology and permeability properties in developing choroid plexus.

ELECTRON microscopical investigations of tight junctions (zonulae occludentes) using marker substances, have indicated that in some tissues these junctions may represent an extracellular pathway for transepithelial movement of certain small solutes1 and also restrict the movement of larger solutes2. The electrical resistance across some epithelial tissues has been used as a measure of the degree of permeability of the tight junctions to ions3, and it was suggested that junctions that are “leaky” to ions are also leaky to small non-electrolytes. Examination of tight junctions with the freeze-fracture technique has revealed the presence of a network of intramembranous fibrils4 or strands5,6. Some evidence suggests that these strands constitute the sealing component of the junction7, and Claude and Goodenough6 correlated the transepithelial permeability of a given epithelium with the number of strands in the tight junction network. Artificial methods of altering transepithelial permeability (hypertonic solutions applied to the mucosal side of the epithelium) have been used to investigate changes in transepithelial electrical resistance8, and freeze-fracture morphology9 or both10. Results from these studies do not consistently support the above correlation. An alternative approach is described here, in which the freeze-fracture technique has been used to study tight junctions of the sheep choroid plexus during foetal development. The results obtained suggest that there is no change in the ultra-structural features of tight junctions which have previously been suggested to correlate with transepithelial permeability, in spite of considerable changes in permeability during the developmental period studied11–13.

Membrane changes associated with assembly of visna virus

Abstract The maturation of visna virus at the surface membranes of sheep choroid plexus cells has been studied by freeze-fracture, deep-etching, surface replication, and scanning EM techniques. The results were integrated with data obtained from thin sections. The first changes detectable in the membranes of infected cells consist of flat regions which lack intramembrane particles and show clusters of globular units on corresponding areas of the surface. Protrusion of the cell membrane, characteristic of the budding process, appears to be initiated after a capsid attaches to these modified membrane regions. During the growth of the viral bud, the areas lacking intramembrane particles, the crescent capsids, and the surface covered with globular units increase in size simultaneously. The surface units become more widely spaced as if they were anchored to the bending capsid. At the end of this development, viruses bud off singly or in rows from the soma and long processes of the infected cells. The capsid detaches from the envelope and forms a central core, the surface units become larger and more closely apposed, and free viruses become smaller than viral buds. Thus, visna virus formation requires insertion and growth of surface units probably representing viral-coded proteins, reorganization of host cell membrane proteins under these units, and, initially, attachment of a coiled capsid to the modified membrane.

Long-term Visna virus infection of sheep choroid plexus cells: initiation and preliminary characterization of the carrier cultures.

Two sheep choroid plexus cell cultures were maintained and propagated for 413 days since being infected with strain K796 visna virus. The majority of the cells in these cultures contained visna virus-specific antigen on days 93 and 105 after infection. Reverse transcriptase-like activity similar to that present in visna virus preparations was obtained from these cultures when very little plaque-forming virus was being synthesized. The persistently infected cultures are resistant to the cytopathic effect which occurs in uninfected cultures upon exposure to visna virus. Persistently infected cells require more time than uninfected cells to become confluent. Less than 0.02 percent of the persistently infected sheep choroid plexus cells form macroscopic colonies within 14 days, whereas 20 to 30 percent of the cells from uninfected cultures form macroscopic colonies within this time.